1. Field of the Invention
This invention is directed to the methods for rapid amplification and detection of actively respiring microorganisms. Such methods are useful in the detection of microbial contaminants in the environment, industrial systems, water purification systems, as well as clinical, food, cosmetic and pharmaceutical samples.
2. Description of the Background
Many U.S. industrial and medical business sectors are at risk for economic loss from microbial contamination. The presence of microorganisms can have a negative impact on business efficiency and productivity. Many bacterial species implicated in human disease as well as those non-pathogenic species that adversely impact industrial processes are ubiquitous in aqueous environments of all types including lakes, rivers, ponds, industrial process waters and even potable water supplies. The total potential economic loss to the U.S. gross domestic product due to microbial contamination has been estimated to be $1-$2 Trillion (THACO Corporation, Independent Market Research, 1993).
Three primary determinants for monitoring microorganism contamination in industrial processes are (i) determination of the quantity of microorganisms that are present, (ii) determination that there are no microorganisms present, and (iii) determination of the specific type (species) of microorganisms that are present.
The classical approach to determinants (i) and (ii) is to culture the sample in question in the presence of selective nutrients and microscopically examine a specimen after staining with specific reagents. Whereas this approach may be satisfactory for some definitive clinical examinations, it is necessary to provide rapid detection, enumeration and identification of microorganisms in industrial and other routine and non-routine medical examinations such as mass casualty or epidemic situations.
Modern methods for microorganism detection and enumeration have focused on the development of more sensitive methods of detecting microorganisms and to a lesser extent upon methods for amplification of the number of microorganisms present in the sample to be analyzed. Some of the technologies in current practice include DNA probes, RNA probes, ATP measurements, immunoassays, enzymatic assays, and respirometic measurements. Many of these tests do not rapidly detect less than 105 cfu/mL or still require complicated or lengthy amplification procedures to increase the concentration of the substrate being detected. Enhancement of the sensitivity of the detection system reduces the threshold concentration of microorganisms to be detected and consequently reduces the time required for amplification. These enhanced assay methods include fluorimetric, radiometric and photometric methods.
For instance, Schapp (U.S. Pat. No. 4,857,652) identified compounds that can be triggered by an activating agent to produce light. This luminescent reaction is used for ultra sensitive detection of phosphatase-linked antibodies and DNA probes. At least one such application of this technology has been commercialized as Photo Gene manufactured by Life Technologies, Inc. (Gaithersburg, Md.). Similarly, Abbas and Eden (U.S. Pat. No. 5,223,402) identify a method that uses 1,2-dioxetane chemiluminescent substrates linked to either alkaline phosphatase or xcex2-D-galactosidase. Theoretically, their method can detect microorganism concentrations as low as 1-100 cfu/mL.
Another strategy for the enhancement of microbial detection is the utilization of fluorescence based detection systems. For example, Fleminger (Eur. J. Biochem. 125:609-15, 1982) used a fluorescent amino benzoyl group that was intra molecularly quenched by a nitrophenylalanyl group. In the presence of bacterial aminopeptidase P, the nitrophenylalanyl group is cleaved and the fluorescence of the sample increases proportionately. A wide variety of other enzymes have been assayed by similar procedures and include hydrolases, carboxypeptidases and endopeptidases. As is the case with the chemiluminescence based assays, fluorescence based assays are susceptible to interferences from chemical quenching agents typical in industrial process waters, require specialized equipment and operator training. Additionally, the fluorescent reagents themselves may be highly toxic and therefore unsuitable for some applications.
Although applicable in certain limited laboratory settings, chemiluminescent methods such as these are susceptible to interference from a variety of chemical quenching agents commonly found in industrial process waters, environmental water sources and biological matrices. Moreover, the methods, as taught in the above referenced patents, require specialized technical training of the user, specialized equipment, multiple steps in the conduct of the assay and enrichment of the microorganism concentration. Taken together, such considerations lengthen the total assay time, raise the capital costs and make this technology unsuitable for high volume, high throughput applications.
Species typing, referred to as determinant (iii) above, not only requires amplification of the microorganisms present, but also requires the selective detection of only the species of interest in the presence of background microflora. The classic approach to species typing is to selectively amplify the presence of the organism of interest through a pre-enrichment step followed by a selective enrichment step using a nutrient specific media followed finally by biochemical or serological confirmation. The time required for these procedures can be as long as six to seven days which is clearly outside the realm of practicality for use in industrial laboratories or high throughput clinical laboratories.
One strategy that has recently been commercialized is the GENE-TRAK(trademark) calorimetric assay (GENE-TRAK Systems, Inc. Framingham, Mass.). This technology attempts to simultaneously exploit an amplification strategy and an enhancement of the detection system""s sensitivity. The approach is an alternative to other approaches that use probes directed against chromosomal DNA. Instead, the GENE-TRAK system targets ribosomal RNA (rRNA) which is present in 1,000-10,000 copies per actively metabolizing cell. A unique homologous series of nucleotides, approximately 30 nucleotides in length and containing a poly-dA tail, is hybridized with the unique rRNA sequence in the target organism. This probe is referred to as the capture probe. A second unique probe of 35-40 nucleotides is labeled at the 3xe2x80x2 and the 5xe2x80x2 ends with fluorescein. This probe is the detector probe and binds to a region of the rRNA adjacent to the capture probe. After hybridization, bound complexes are captured on a solid support coated with poly-dT which hybridizes with the poly-dA tail of the capture probe. The rRNA-detector probe complex is detected with polyclonal anti-fluorescein antibody conjugated to horseradish peroxidase. This complex is then reacted with the enzyme substrate, hydrogen peroxide, in the presence of tetramethylbenzidine. The blue color that develops is proportional to the amount of rRNA captured.
Blackburn reviewed the development of rapid alternative methods for microorganism typing as it pertains to the food industry (C. de W. Blackburn, xe2x80x9cRapid and alternative methods for the detection of salmonellas in foods,xe2x80x9d Journal of Applied Bacteriology, 75:199-214, 1993). Therein, Blackburn describes several techniques for detection of Salmonella that rely upon a selective pre-enrichment and enrichment approach to amplification, the best of which still required approximately six hours before detectable levels of Salmonella were present. Enhanced detection methods were also reviewed and included measurements of metabolism, immunoassays, fluorescent-antibody staining, enzyme immunoassay, immunosensors, bacteriophages and geneprobes. Analysis times could be reduced to as short as 20 minutes; the detection limits were about 105 cfu (Blackburn et al., xe2x80x9cSeparation and detection methods for salmonellas using immunomagnetic particles,xe2x80x9d Biofouling 5:143-156, 1991). Similarly the detection limits could be reduced to as low as 1-10 cfu, however the enrichment protocols required 18-36 hours. In all cases, the described methods provided detection limits that were either too high or analysis times that were too long to be practical for application to industrial processes and high volume, high throughput clinical situations.
There have been numerous approaches to microorganism detection and typing. For instance, U.S. Pat. No. 4,376,110 describes a solid-phase immunoassay employing a monoclonal capture antibody and a labeled secondary antibody. Alternatively, U.S. Pat. No. 4,514,508 utilizes labeled complement and U.S. Pat. Nos. 4,281,061; 4,659,678; and 4,547,466 describe other immunological based variants. All of these methods require from 103 to 107 cfu/mL to reliably detect the target microorganism. Necessarily, additional enrichment steps are required which add several hours to days to the assay procedure.
Various enrichment techniques are described as well. For instance Valkirs (U.S. Pat. No. 4,727,019) and Hay-Kaufman (U.S. Pat. No. 4,818,677) describe flow-through devices to capture cells and in situ immunoassay to detect the presence of the target organism. Schick (U.S. Pat. No. 4,254,082) describes an ion exchange particle system for capturing the target organism and Chau (U.S. Pat. No. 4,320,087) describes an activated charcoal coated bead capture device. All of these devices suffer several limitations such as small volume capacities, fouling from the presence of particulates in the sample or nonspecificity of the capture process and are as a consequence unsatisfactory for large volume, high throughput industrial and clinical applications.
While it has been shown that a number of technologies have been developed for a variety of applications, it is also clear that there continues to be a need for the development of simple, sensitive, rapid, inexpensive and reliable detection systems with applicability to the broad scope of industrial and clinical processes.
The present invention overcomes the problems and limitations of current methods for rapid detection and enumeration of microorganisms such as bacteria by providing methods for amplification of the presence of microorganisms in a sample without reliance upon pre- or post-enrichment processes. The present invention also provides for the highly sensitive, quantitative enumeration of total viable microorganisms with a detection limit less than 1000 cfu/mL and an analysis time less than two hours. The present invention is amenable to the analysis of clinical, food, cosmetic, pharmaceutical, industrial and environmental samples without regard to the sample matrix. It is clear to one skilled in the art that the net effect of these embodiments provides a sensitive and fast microorganism-detection technology.
Advantages of the invention are set forth, in part, in the description which follows and, in part, will be obvious from this description and may be learned from the practice of this invention.